Autocrine/paracrine regulatory mechanisms are believed to play a role in the pathophysiology of several hematologic malignancies. Evidence is accumulating that various growth factors, cytokines, and chemokines are expressed and secreted by normal early and differentiated hematopoietic cells and thus could also regulate normal hematopoiesis in an autocrine/paracrine manner. In this review we summarize recent advances in identification and understanding of the role of autocrine/paracrine axes in the growth of both malignant and normal human hematopoietic cells. Better understanding of intercellular crosstalk operating in normal and pathological states and the mechanisms regulating synthesis of these endogenously produced factors (potential targets for various pharmacological approaches) may allow us to improve antileukemia treatments, undertake more efficient ex vivo stem cell expansion, and develop other therapeutic strategies.

The development of hematopoietic cells is regulated by hematopoietic growth factors, cytokines, and chemokines secreted by: A) hematopoietic accessory cells (e.g., fibroblasts, endothelial cells, macrophages, and osteoblasts) residing in the bone marrow (BM) microenvironment; B) various nonhematopoietic organs (e.g., liver, kidney), and C) normal T lymphocytes (e.g., as part of Th1- and Th2-mediated responses) [1-8]. All these factors together modulate the biology of early hematopoietic cells present in the BM niches (Fig. 1).

Figure Figure 1.. Early hematopoietic cells which reside in BM niches are under the influence of various growth factors.Cytokines and chemokines are secreted by stromal fibroblasts (1) and endothelium (2) or are derived from other organs (kidney, liver, etc.) and arrive via the blood stream (3), accessory cells (4), differentiating hematopoietic cells (5), osteoblasts (6), and, in an autocrine manner, early hematopoietic cells (7). Interaction of hematopoietic cells with adhesion receptors expressed in the hematopoietic microenvironment may also play a regulatory role (8).

The existence of several putative autocrine/paracrine loops was first demonstrated in human hematologic malignancies [9-14]. More recently, we and others used reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate the presence of mRNA for several growth factors, cytokines, and chemokines in normal human CD34+ cells [15-18], megakaryocytes [19-20], and other BM mononuclear cells (BMMNC) [21]. We also demonstrated that various growth factors, cytokines, and chemokines are secreted by normal human early and differentiated hematopoietic cells and thus could regulate hematopoiesis in an autocrine/paracrine manner [17, 22-25]. These recent observations may change our traditional view of regulation of the early stages of hematopoietic development.

Here we review the evidence supporting the role of autocrine/paracrine axes in the growth of malignant as well as normal human hematopoietic cells. We will focus on the expression of these regulators by hematopoietic cells as well as on the biological consequences of their endogenous secretion.

Factors endogenously secreted by leukemic cells may contribute to leukemogenesis, and cells that synthesize a growth factor and simultaneously express on their surface its corresponding receptor may proliferate by a self-stimulatory or autocrine mechanism. If the factor is secreted by the cells and activates surface receptors, it operates via a “public autocrine loop” [9-13]. In contrast, if the mitogenic signal is generated without evidence of factor secretion, the growth factor may activate a corresponding receptor intracellularly via a “private autocrine loop” [13, 27-29]. In this latter situation a ligand-receptor interaction occurs in the intracellular compartment.

It is generally accepted that cells that secrete active growth factors, and thus depend on “public autocrine loops,” proliferate at high density in cultures and that their growth is inhibited in the presence of antibodies that neutralize the putative stimulator of autocrine growth. In contrast, the growth of cells in which the autocrine factor interacts with the corresponding receptor intracellularly is neither density-dependent nor inhibited by neutralizing antibodies.

There is growing evidence that both mechanisms may affect malignant hematopoiesis. The existence of “public autocrine loops” has been described in cells derived from the blood of patients with acute myelogenous leukemia ([AML] GM-CSF, G-CSF, macrophage-CSF [M-CSF], and interleukin 1β [IL-1β] [9-11]), chronic myelogenous leukemia (IL-3, G-CSF, M-CSF, and IL-1β) [12, 30], and multiple myeloma (IL-6 and kit ligand [KL]) [31]. A “private autocrine loop” has been shown to operate in some human primary AML cells as well as murine experimental models [27-29]. It has been reported that IL-3-dependent murine cell lines (32D and FDC-P1) proliferate when IL-3 is expressed in these cells even as an intracellular, nonsecretable protein.

However, experiments in transgenic mice expressing IL-3, GM-CSF, and IL-6 in hematopoietic organs have demonstrated that autocrine/paracrine expression of these factors results in a chronic hyperproliferative state but not acute neoplastic transformation [14]. This supports the notion that several circumstances are necessary for malignant transformation, and autocrine or paracrine production of hematopoietic growth factors or cytokines alone is not sufficient for leukemogenesis. Other “second hit”-type mutations in oncogenes and/or anti-oncogenes are necessary to transform cells. In this context factors secreted by leukemic cells may, for example, inhibit apoptosis of leukemic blasts, allowing these cells to accumulate and become targets for a “second hit,” activating oncogenes and/or inactivating anti-oncogenes.

Factors secreted by leukemic cells may also act in a paracrine manner on surrounding cells; for example, stromal or endothelial cells can be stimulated by one factor to produce other multiple hematopoietic growth factors which in turn may affect growth of leukemic blasts. Certain factors secreted by leukemic cells may stimulate expansion and activation of endothelium (e.g., vascular endothelial growth factor [VEGF], fibroblast growth factor [FGF-2]) or BM stromal fibroblasts (platelet-derived growth factor) [32-36]. Subsequently, such expanded and activated endothelium and/or stroma could provide a more permissive environment for leukemic growth [32-36]. Expanded endothelium resulting from paracrine stimulation by VEGF or FGF-2 (both shown to be secreted by leukemic blasts) has been postulated to play an important role in the pathogenesis and progression of human acute leukemias [32, 35, 36], chronic myeloid leukemia [33, 34], myeloproliferative disorders [37], and multiple myelomas [38]. These paracrine effects of leukemic cells on marrow endothelium closely resemble the expansion of endothelium and neo-angiogenesis observed in solid tumor tissues [39-42].

We now know that several growth factors, cytokines, and chemokines are expressed and secreted by normal human early and differentiated hematopoietic cells and thus may regulate hematopoiesis in an autocrine/paracrine manner. For example, it has been reported that normal human CD34+ cells express mRNA for IL-1β [15] and tranforming growth factor (TGF)-β1 [16, 43-45]. Similarly, we found that both CD34+ and CD34+kit+ cells enriched in stem/progenitor cells express mRNA for KL and FLT-3 [17]. Recently, it was found that CD34+ cells express mRNA for erythropoietin (EPO) [46-48] and stromal-derived factor (SDF)-1 [49]. Similarly, mRNA for IL-1β, IL-6, GM-CSF, and tumor necrosis factor (TNF)-α was found to be expressed in normal human megakaryoblasts [19, 20] and mRNA for EPO in normal erythroblasts [46]. Finally, several cytokines and growth factors were found to be expressed at the mRNA level in peripheral blood-derived MNC [21].

To extend these studies we isolated highly purified CD34+ cells, myeloblasts, megakaryoblasts, and erythroblasts and found these cells expressed mRNA for various growth factors, cytokines, and chemokines (Table 1) [23, 24]. The hematopoietic growth factors we selected are known to be regulators of mesodermal development and activate receptors possessing intrinsic tyrosine kinase activity. We found that human BM-derived CD34+ cells express mRNA for KL, FLT-3, FGF-2, VEGF, hepatocyte growth factor (HGF), and insulin-like growth factor (IGF)-1. We also observed that normal human BM CD34+ cells express mRNA for growth factors and cytokines that may stimulate cell proliferation (thrombopoietin [TPO] and IL-1) or alternatively inhibit it (TNF-α, interferon-α, and Fas-L). On the other hand, mRNA for M-CSF and nerve growth factor-β was not expressed in these cells. We were also not able to detect mRNA for IL-3, IL-6, EPO, G-CSF, and GM-CSF. However, evaluating whether these cells express mRNA for various chemokines, we found mRNAs for macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES, MCP-3, MCP-4, IL-8, IP-10, MDC, and PF-4 in highly purified human BM CD34+ cells. Similarly, mRNA for several hematopoietic regulators was found to be expressed in colony-forming unit-granulocyte, macrophage (CFU-GM)-, BFU-E-, and CFU-megakaryocyte (Meg)-derived cells (Table 1).

Table Table 1.. RT-PCR analysis (30 cycles) of highly purified CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts for expression of various growth factors, cytokines, and chemokines. Cells from three independent donors were analyzed with similar results.

CD34+ BM

CFU-GM-derived

BFU-E-derived

CFU-Meg-derived

KL

+

+

+

+

FLT-3

+

+

+

–

M-CSF

–

+

+

+

FGF-2

+

+

+

+

VEGF

+

+

+

+

HGF

+

–

+

+

IGF-I

+

–

+

+

NGF-β

–

–

–

–

IL-1

+

–

–

–

IL-3

–

–

–

–

IL-6

–

–

–

–

EPO

–

–

–

–

TPO

+

+

+

+

G-CSF

–

–

–

–

GM-CSF

–

–

–

+

TNF-α

+

+

+

+

IFN-α

+

+

+

+

Fas-L

+

+

+

+

MIP-1α

+

+

+

+

MIP-1β

+

+

–

+

RANTES

+

+

–

+

MCP-1

–

–

–

–

MCP-2

–

+

+

+

MCP-3

+

+

–

+

MCP-4

+

+

+

+

IL-8

+

–

–

+

IP-10

+

+

+

+

MDC

+

–

–

–

SDF-1

–

–

–

–

PF-4

+

+

+

+

Furthermore, the micro-array strategy, a powerful tool to screen mRNAs expressed in human cells, has also been successfully employed to screen mRNA molecules expressed in normal and malignant human hematopoietic cells [25, 50].

Although the expression of various hematopoietic regulators has been demonstrated at the mRNA level, it has not been clear whether these regulators are expressed at the protein level and secreted by early normal human hematopoietic cells. Functional studies performed with anti-TGF-β-blocking monoclonal antibodies (mAbs) [16], as well as studies of downregulation of mRNA encoding KL and FLT-3 using antisense oligomers [17], supported the notion that these factors are expressed at the protein level and modulate the biology of CD34+ cells. Moreover, in a recent study using immunohistochemistry, it has been demonstrated that normal human CD34+ cells express IL-8 and IL-1 proteins [18].

Table Table 2.. Various growth factors, cytokines, and chemokines detected by ELISA in media conditioned by normal human hematopoietic cells. Cells from three to five independent healthy donors were analyzed with similar results.

Moreover, using the sensitive ELISA we also found that highly purified human CD34+ cells secrete chemokines such as MIP-1α, MIP-1β, RANTES, IL-8, and PF-4. The potential biological significance of these chemokines is intriguing. Endogenously secreted chemokines could: A) attract other cells; B) stimulate angiogenesis, and C) regulate cell migration and adhesion. We also observed that several of these chemokines are secreted by CFU-GM-, BFU-E-, and CFU-Meg cells derived from ex vivo-expanded CD34+ cells.

While we were able to detect mRNA for different growth factors and cytokines in normal early human hematopoietic cells (Table 1), it is not surprising that many of these factors were not detectable at the protein level in media conditioned by these cells (Table 2).

Several methods are currently available to study the biological function of putative autocrine regulatory loops. Generally, the function of these loops may be perturbed by using: A) mAbs against ligands and their corresponding receptors [16]; B) antisense oligodeoxynucleotides against mRNA encoding factors or their receptors [17]; C) modified ligands or synthetic molecules that block surface receptors [52]; D) an intracrine strategy [53], and finally, E) by “homologous-recombination” which allows “knock-out” of appropriate genes involved in the particular axes [54]. Generally, blocking-mAbs are more suitably used for investigating public, and antisense oligomers for studying private, autocrine regulatory loops.

Biological Implications of Endogenous Expression and Secretion of Hematopoietic Regulators

Cells that express and synthesize a growth factor or growth-regulating cytokine and simultaneously express the corresponding receptor for this factor have the potential for self-stimulatory or autocrine growth. Nevertheless, endogenously secreted factors, in addition to their influence on proliferation, may also have other biological functions stimulating cells in an autocrine or paracrine manner, and the proper biological combination of these various factors may be necessary to provide stimulatory signals and gradients to the cells. Hence various scenarios in which these mechanisms operate are possible. For example, endogenously secreted factors may serve as chemoattractants for other hematopoietic cells (accessory cells, facilitating cells, etc.), stimulate the secretion of other regulatory molecules, modulate the expression of adhesion molecules on the cell surface (and thus regulate their adhesion and homing), and/or influence the survival of the hematopoietic cells.

It has been reported that neutralizing (by mAb) of TGF-β1, which is endogenously secreted by cord blood-derived CD34+ cells, increased the formation of hematopoietic colonies [16]. Moreover, it has been shown that blocking of the autocrine inhibitory TGF-β1-TGF-β1R axis improved ex vivo expansion of these cells [16, 45]. Hence inhibition of the autocrine negative regulatory axes could provide a useful strategy for optimizing the ex vivo expansion of early hematopoietic cells. Since the number of progenitor cells in cord blood is limited, this strategy could significantly expand their availability for clinical transplantation. Autocrine negative regulatory axes could be perturbed by mAbs or antisense oligodeoxynucleotides targeted against negative regulators and/or their corresponding receptors.

Similarly, by employing appropriate antisense oligomers targeted against mRNA encoding several tyrosine kinase receptors, we were able to show that perturbation of expression of receptors for KL and FLT-3 [17], but not VEGF, FGF-2, or HGF [33, 55, 56], affects the proliferation and survival of human CD34+ cells, a finding which suggests that both KL and FLT-3 may operate as positive autocrine regulators in these cells [17]. In agreement with these observations, other investigators have also recently suggested the secretion of KL by normal CD34+ cells, having found that kitlow/- CD34+ cells began to proliferate after transduction with kit cDNA, even when exogenous KL was absent from the culture medium [57]. Moreover, since human CD34+ cells secrete TPO and express c-mpl [58, 59], a putative role for c-mpl-TPO in autocrine regulation of CD34+ cells has also been postulated but requires further investigation. Similarly, as human CD34+ cells express CD95 (Apo/Fas) and TNF-R (p55 and p7E) (Fig. 2), and as we found that mRNA for Fas-L and TNF-α is expressed in these cells (Table 1), we suggest that two other negative regulatory axes (Fas-L-CD95 and TNF-α-TNF-R), in addition to the TGF-β1-TGF-βR axis [16, 43-45, 60], may operate in the early stages of hematopoiesis.

Figure Figure 2.. Expression of TNF-R1 (p55-CD120a), TNF-R2 (75p-CD120b), and CD95 (APO-1/Fas) on BM CD34+BMMNC.BMMNC were isolated from BM aspirates of healthy donors by Ficoll-gradient centrifugation and stained with mAb against CD34 antigen (fluorescein isothiocyanate) and TNF-R1, TNF-R2, and CD95 receptors (phycoerythrin [PE]). A) Forward and side scatter analysis of CD34+cells isolated from BMMNC isolated from BM CD34+cells. Lymphocyte region is defined by R1. B) Purity of CD34+cells is shown by immunostaining with PE-anti-CD34 mAb. C) Analysis of CD34+cells labeled with PE-anti-TNF-R1 mAb. D) Analysis of CD34+cells labeled with PE-anti-TNF-R2 mAb. E) Analysis of CD34+cells labeled with PE-anti-CD95 mAb. All the mAbs were from Pharmingen (San Diego, CA). Data from at least five different donors were analyzed with similar results. Data from a representative donor are presented.

Although the precise role in normal hematopoiesis of the numerous endogenously secreted hematopoietic regulators requires further investigation, it is worth noting that even if endogenous factors are secreted at very low levels, they may be much more biologically effective than corresponding recombinant proteins employed at similar doses [61]. Thus the endogenously secreted factors we identified (and likely those as yet undiscovered) may play a key role in the biology of early hematopoietic cells in vivo. In vitro, where recombinant molecules are used, this effect will likely differ. As an example, autocrine TGF-β1 secretion in human hematopoietic progenitors upregulates bcl-2 expression and plays an important role in regulating the survival and differentiation of primitive proliferating hematopoietic progenitors by cell cycle-independent mechanisms [44], whereas exogenously added TGF-β1 affects the cell cycle and inhibits proliferation of these cells [16, 45].

Studying autocrine/paracrine mechanisms regulating normal human hematopoiesis we observed that CD34+ cells cultured ex vivo in conditioned media harvested from other CD34+ cells survive better. This suggested that in the mixture of various factors secreted by these cells, the effect of antiapoptotic factors prevailed [23, 24]. We also observed that conditioned media harvested from CD34+ cells chemoattract other CD34+ cells as well as normal human lymphocytes [23, 24]. Since CD34+ cells do not secrete SDF-1 (Table 2), this observation suggests that another important as yet unidentified chemoattractant for human CD34+ cells exists and is endogenously secreted by them [23, 24]. The fact that CD34+ cells also attract lymphocytes suggests that this mechanism could be employed to attract certain subpopulations of lymphoid cells or facilitating cells which perhaps may play a regulatory role in the development of early hematopoietic cells [24]. We also reported that conditioned media harvested from CD34+ cells containing VEGF, FGF-2, HGF, and IL-8 stimulated proliferation of human endothelium (Table 2). Thus, crosstalk between hematopoietic cells and endothelium is not only evident in malignant hematopoiesis but also during normal hematopoiesis [32, 35, 40, 42, 53, 62]. Further, we found that some of the identified β-chemokines secreted by CD34+ cells that bind to CCR5, such as MIP-1α, MIP-1β, and RANTES, may interfere with the infectability of these cells by R5 HIV (macrophagotropic human immunodeficiency virus) by competing with the virus for binding to CCR5 [22, 51]. Similarly RANTES endogenously secreted by human megakaryoblasts and megakaryocytes protects these cells from infection by R5 HIV. Thus, since endogenously secreted β-chemokines could contribute to AIDS-free status, we predict that therapeutic agents that may stimulate the endogenous synthesis of these chemokines in human hematopoietic cells could protect them from HIV infection, and we recommend their development in the future.

In addition to cytokines, growth factors, and chemokines, there are many other proteins that are secreted by different subsets of normal human hematopoietic cells and may modulate hematopoiesis. The most important candidates are matrix metalloproteinases [63], tissue inhibitors of metalloproteinases, morphogenetic proteins, complement proteins (C3), soluble receptors (sFas, sgp130, sTNF-R1, sTNF-R2, uPAR, etc.), and soluble adhesion molecules (cCD14, sCD31, sL-selectin, sP-selectin, etc.). These proteins are detectable by sensitive ELISA, zymography, reverse zymography, Western-blotting, and immunocytochemistry.

In the last few years, a powerful proteomics strategy has been developed, and we are employing it to screen conditioned media harvested from CD34+ cells, as well as lineage-expanded BFU-E-, CFU-GM-, and CFU-Meg-derived cells, for the presence of various proteins. First, the conditioned media are concentrated and then the proteins present in these concentrates are fractionated, separated by two-dimensional electrophoresis, isolated from the gels, and subjected to mass-spectrophotometry. Finally, the identified peptides are screened against a proteomics database [64]. This strategy is helping us to identify other hematopoietic regulators secreted by normal human hematopoietic cells.

In conclusion, evidence is accumulating that normal human hematopoietic cells (BM or PB CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts) secrete numerous growth factors, cytokines, and chemokines which contribute to intercellular crosstalk networks and regulate the various stages of hematopoiesis. We believe that these observations direct us towards a new area of investigation into human hematopoiesis.